Funktionelle und strukturelle Eigenschaften des mitochondrialen Phosphatcarriers aus Saccharomyces cerevisiae

Schroers, A.

Book/Report

FZJ-2019-03751

Funktionelle und strukturelle Eigenschaften des mitochondrialen Phosphatcarriers aus Saccharomyces cerevisiae

Schroers, A. (Corresponding author)FZJ*

1998
Forschungszentrum Jülich, Zentralbibliothek, Verlag Jülich

Jülich : Forschungszentrum Jülich, Zentralbibliothek, Verlag, Berichte des Forschungszentrums Jülich 3546, 83 p. (1998)

Please use a persistent id in citations: http://hdl.handle.net/2128/22410

Report No.: Juel-3546

Abstract: Uptake of inorganic phosphate into mitochondria is essential for energy metabolism. The import of phosphate into the mitochondrial matrix is mediated by the phosphate carrier (PIC), a 32 kDa protein which catalyzes the electroneutral import of phosphate (H$_{2}$PO$_{4}$) compensated by the antiport of a hydroxyl ion. We have expressed wild type and mutant PIC from yeast mitochondria in Escherichia coil inclusion bodies. PIC was solubilized from inclusion bodies, purified and reconstituted into proteoliposomes in a functionally active form. The mitochondrial phosphate carrier (PIC) belongs to the mitochondrial carrier family (MCP). Members of this family comprise six transmembrane segments and have been shown to exist as dimers in the solubilized state. Beneath the strictly coupled transport functions of the PIC there is an uncoupled transport mode which can be observed after modification of the carrier with HgCl$_{2}$. After the analysis of mutants in which the three cysteines of the wt-PIC were exchanged with serine, the modification of the cystein residue in position 28 turned out to be the trigger for uncoupling. By the introduction of amino acids different in size and in charge in position of cystein 28 a positive charge was found to be responsible for the induction of the uncoupled efflux mode. In order to investigate a functional necessity of the dimerization and the molecular me chanism of coupling, we have developed a method to isolate tagged proteins as monomers, to construct defined dimers from these monomers, and to isolate specific dimeric forms. By functional analysis of heterodimers consisting of functionally different monomers the following conclusions can be drawn: (i) Dimerization is essential for functionality, (ii) formed dimers are stable, (iii) there is functional crosstalk between the subunits of the dimer. (iv) modification (mutation) of one monomer is sufficient for uncoupling. Furthermore the functional analysis of heterodimers with different amino acids in position 28 provided evidence for one single pathway within the functional active dimer.

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